mda-9/Syntenin protein positively regulates the activation of Akt protein by facilitating integrin-linked kinase adaptor function during adhesion to type I collagen

The integrin-linked kinase (ILK)-PINCH1-α-parvin (IPP) complex functions as a signaling platform for integrins that modulates various cellular processes. ILK functions as a central adaptor for the assembly of IPP complex. We report here that mda-9/syntenin, a positive regulator of cancer metastasis, regulates the activation of Akt (also known as protein kinase B) by facilitating ILK adaptor function during adhesion to type I collagen (COL-I) in human breast cancer cells. COL-I stimulation induced the phosphorylation and plasma membrane translocation of Akt. Inhibition of mda-9/syntenin or expression of mutant ILK (E359K) significantly blocked the translocation of both ILK and Akt to the plasma membrane. mda-9/syntenin associated with ILK, and this association was increased at the plasma membrane by COL-I stimulation. Knockdown of mda-9/syntenin impaired COL-I-induced association of ILK with Akt and plasma membrane targeting of ILK-Akt complex. These results demonstrated that mda-9/syntenin regulates the activation of Akt by controlling the plasma membrane targeting of Akt via a mechanism that facilitates the association of Akt with ILK at the plasma membrane during adhesion to COL-I. On a striking note, inhibition of mda-9/syntenin impaired COL-I-induced plasma membrane translocation of the IPP complex and assembly of integrin β1-IPP signaling complexes. Thus, our study defines the role of mda-9/syntenin in ILK adaptor function and describes a new mechanism of mda-9/syntenin for regulation of cell migration.     

Expression of insulin‐like growth factor‐1 receptor in metastatic uveal melanoma and implications for potential autocrine and paracrine tumor cell growth

We investigated the importance of the insulin‐like growth factor‐1 receptor (IGF‐1R) in hepatic metastases of uveal melanoma. The expression pattern of IGF‐1R in archival tissue samples of hepatic metastasis from 24 patients was analyzed by immunohistochemistry. All the samples of hepatic metastases stained positive for IGF‐1R. To investigate the biological role of IGF‐1R on the growth of metastatic uveal melanoma, a long‐term cell line obtained from a hepatic metastasis (TJU‐UM001) was evaluated. TJU‐UM001 expressed cell surface IGF‐1R (>90%) and proliferated in response to exogenous and endogenous insulin‐like growth factor‐1 (IGF‐1). Correlatively, anti‐IGF‐1R antibody completely blocked IGF‐1‐induced growth of TJU‐UM001 cells. IGF‐1 preferentially induced phosphorylation of Akt (S473) in quiescent TJU‐UM001 cells, and this was blocked by anti‐IGF‐1R antibody. This study suggests that autocrine and paracrine mechanisms underlie IGF‐1‐induced growth of metastatic uveal melanoma and underscore the potential benefit of IGF‐1 or IGF‐1R antagonism in treatment for metastatic uveal melanoma.     

Decreased expression of Apaf-1 with progression of melanoma

Defects in apoptotic system may contribute in the pathogenesis and resistance of malignant melanoma cells to chemotherapy. Apoptotic protease-activating factor-1 (Apaf-1) is a cell death effector that acts with cytochrome c and caspase-9 to mediate apoptosis. Recently it was shown that metastatic melanomas often lose Apaf-1 and are concomitantly resistant to apoptosis. It is not known, however, whether Apaf-1 protein is lost during melanoma progression from localized to metastatic tumor. To this end, we evaluated Apaf-1 protein expression by immunohistochemistry in 10 cases of human nevi, 11 melanomas in situ, 26 primary melanomas and 15 metastases. Significant decreases in Apaf-1 expression was observed when comparing nevi and melanomas (chi-square = 33.719; P < 0.0001). Moreover, primary melanomas with greater tumor thickness showed lesser expression of Apaf-1 (chi-square = 16.182; P < 0.003). Intriguingly, we were unable to detect Apaf-1 expression in lesions of metastatic melanomas. These data demonstrated that there is an inverse correlation between Apaf-1 expression and pathologic stage of melanoma. This suggests that the decreased expression of Apaf-1 seen in correlation with melanoma progression renders melanoma more resistant to chemotherapy.     

Decreased expression of Apaf‐1 with progression of melanoma

Defects in apoptotic system may contribute in the pathogenesis and resistance of malignant melanoma cells to chemotherapy. Apoptotic protease‐activating factor‐1 (Apaf‐1) is a cell death effector that acts with cytochrome c and caspase‐9 to mediate apoptosis. Recently it was shown that metastatic melanomas often lose Apaf‐1 and are concomitantly resistant to apoptosis. It is not known, however, whether Apaf‐1 protein is lost during melanoma progression from localized to metastatic tumor. To this end, we evaluated Apaf‐1 protein expression by immunohistochemistry in 10 cases of human nevi, 11 melanomas in situ, 26 primary melanomas and 15 metastases. Significant decreases in Apaf‐1 expression was observed when comparing nevi and melanomas (chi‐square = 33.719; P < 0.0001). Moreover, primary melanomas with greater tumor thickness showed lesser expression of Apaf‐1 (chi‐square = 16.182; P < 0.003). Intriguingly, we were unable to detect Apaf‐1 expression in lesions of metastatic melanomas. These data demonstrated that there is an inverse correlation between Apaf‐1 expression and pathologic stage of melanoma. This suggests that the decreased expression of Apaf‐1 seen in correlation with melanoma progression renders melanoma more resistant to chemotherapy.     

CXC chemokine receptor 4 is expressed in uveal malignant melanoma and correlates with the epithelioid-mixed cell type

PURPOSE: Although relatively rare, uveal melanoma is the most common ocular tumor of adults. Up to half of uveal melanoma patients die of metastatic disease. CXCR4, a chemokine receptor, is a prognostic factor in cutaneous melanoma involved in angiogenesis and metastasis formation. The aim of this study was to evaluate the expression of CXCR4 in uveal melanoma. METHODS: CXCR4 was detected by immunohistochemistry in 44 samples of uveal melanoma. Staining was categorized into three semiquantitative classes based on the rate of stained (positive) tumor cells: absence of staining, <50% of cell (+) and >50% (++). Correlations between CXCR4 expression, data on patient and tumor features were studied by contingency tables and the chi2 test. Time-to-event curves were studied using the Kaplan-Meier method. Univariate analysis was performed using the log-rank test. Ninety-five percent confidence intervals (95% CI) of hazard ratios were also reported. RESULTS: Staining for CXCR4 protein was absent in 18 tumors (40.9%), present in <50% of cells in 19 (43.2%) and in >50% of cells in 7 (15.9%) tumors. CXCR4 expression correlated to the epithelioid-mixed cell type (P=0.030). No statistically significant relation emerged between CXCR4 expression, largest tumor diameter (LTD) and extracellular matrix patterns as evaluated through histological patterns stained with periodic acid-Schiff (PAS). Events occurred in 2 out of 18 patients (11.1%) with negative tumors (2 deaths), in 3 out of 19 patients (15.8%) with <50% of positive tumor cells (2 deaths and 1 occurrence of metastases) and in 1 out of 7 patients (14.3%) with >50% of positive tumor cells (1 occurrence of metastases). The cell type (P=0.0457) but not CXCR4 showed prognostic value at univariate analysis. CONCLUSION: This study shows that CXCR4 is commonly expressed in uveal melanoma and correlates with cell type a well-established prognostic factor.     

ABCB1 identifies a subpopulation of uveal melanoma cells with high metastatic propensity

Metastasis of tumor cells to distant organs is the leading cause of death in melanoma. Yet, the mechanisms of metastasis remain poorly understood. One key question is whether all cells in a primary tumor are equally likely to metastasize or whether subpopulations of cells preferentially give rise to metastases. Here, we identified a subpopulation of uveal melanoma cells expressing the multidrug resistance transporter ABCB1 that are highly metastatic compared to ABCB1(-) bulk tumor cells. ABCB1(+) cells also exhibited enhanced clonogenicity, anchorage-independent growth, tumorigenicity and mitochondrial activity compared to ABCB1(-) cells. A375 cutaneous melanoma cells contained a similar subpopulation of highly metastatic ABCB1(+) cells. These findings suggest that some uveal melanoma cells have greater potential for metastasis than others and that a better understanding of such cells may be necessary for more successful therapies for metastatic melanoma.     

Ionizing radiation enhances adenoviral vector expressing mda-7/IL-24-mediated apoptosis in human ovarian cancer

Ovarian cancer is the fifth most common cause of cancer-related death in women. Current interventional approaches, including debulking surgery, chemotherapy, and/or radiation have proven minimally effective in preventing the recurrence and/or mortality associated with this malignancy. Subtraction hybridization applied to terminally differentiating human melanoma cells identified melanoma differentiation associated gene-7/interleukin-24 (mda-7/IL-24), whose unique properties include the ability to selectively induce growth suppression, apoptosis, and radiosensitization in diverse cancer cells, without causing any harmful effects in normal cells. Previously, it has been shown that adenovirus-mediated mda-7/IL-24 therapy (Ad.mda-7) induces apoptosis in ovarian cancer cells, however, the apoptosis induction was relatively low. We now document that apoptosis can be enhanced by treating ovarian cancer cells with ionizing radiation (IR) in combination with Ad.mda-7. Additionally, we demonstrate that mda-7/IL-24 gene delivery, under the control of a minimal promoter region of progression elevated gene-3 (PEG-3), which functions selectively in diverse cancer cells with minimal activity in normal cells, displays a selective radiosensitization effect in ovarian cancer cells. The present studies support the use of IR in combination with mda-7/IL-24 as a means of augmenting the therapeutic benefit of this gene in ovarian cancer, particularly in the context of tumors displaying resistance to radiation therapy.     

Expression Profiling of Primary and Metastatic Ovarian Tumors Reveals Differences Indicative of Aggressive Disease

The behavior and genetics of serous epithelial ovarian cancer (EOC) metastasis, the form of the disease lethal to patients, is poorly understood. The unique properties of metastases are critical to understand to improve treatments of the disease that remains in patients after debulking surgery. We sought to identify the genetic and phenotypic landscape of metastatic progression of EOC to understand how metastases compare to primary tumors. DNA copy number and mRNA expression differences between matched primary human tumors and omental metastases, collected at the same time during debulking surgery before chemotherapy, were measured using microarrays. qPCR and immunohistochemistry validated findings. Pathway analysis of mRNA expression revealed metastatic cancer cells are more proliferative and less apoptotic than primary tumors, perhaps explaining the aggressive nature of these lesions. Most cases had copy number aberrations (CNAs) that differed between primary and metastatic tumors, but we did not detect CNAs that are recurrent across cases. A six gene expression signature distinguishes primary from metastatic tumors and predicts overall survival in independent datasets. The genetic differences between primary and metastatic tumors, yet common expression changes, suggest that the major clone in metastases is not the same as in primary tumors, but the cancer cells adapt to the omentum similarly. Together, these data highlight how ovarian tumors develop into a distinct, more aggressive metastatic state that should be considered for therapy development.     

PD‐L1 expression in melanoma shows marked heterogeneity within and between patients: implications for anti‐PD‐1/PD‐L1 clinical trials

This study evaluated the expression of PD‐L1 in immunotherapy‐naïve metastatic melanoma patients to determine longitudinal intrapatient concordance and correlate PD‐L1 status with clinicopathologic characteristics and outcome. PD‐L1 expression was assessed by immunohistochemistry in 58 patients (43 primary tumors, 96 metastases). Seventy‐two percent of patients had at least one specimen expressing PD‐L1 in ≥1% of tumor cells. Median positive tumor cell count overall was low (8% in nonzero specimens). PD‐L1 expression was frequently discordant between primary tumors and metastases and between intrapatient metastases, such that 23/46 longitudinal patient specimens were discordant. PD‐L1 was associated with higher TIL grade but not with other known prognostic features. There was a positive univariate association between PD‐L1 expression in locoregional metastases and melanoma‐specific survival, but the effect was not observed for primary melanoma. In locoregional lymph node metastasis, PD‐L1+/TIL+ patients had the best outcome, and PD‐L1+/TIL? patients had poor outcome.     

Regulation of protective autophagy in anoikis-resistant glioma stem cells by SDCBP/MDA-9/Syntenin

Glioblastoma multiforme (GBM) is a frequent and aggressive glial tumor, containing a small population of therapy-resistant cells, glioma stem cells (GSCs). Current dogma suggests that tumors regrow from GSCs, and these cells contribute to therapy resistance, poor prognosis, and recurrence; highlighting the importance of GSCs in glioma pathophysiology and therapeutic targeting. Macroautophagy/autophagy-based cellular homeostasis can be changed from pro-survival to pro-cell death by modulating SDCBP/MDA-9/Syntenin (syndecan binding protein)-mediated signaling. In nonadherent conditions, GSCs display protective autophagy and anoikis-resistance, which correlates with expression of SDCBP/MDA-9/Syntenin. Conversely, SDCBP/MDA-9/Syntenin silencing induces autophagic death in GSCs, indicating that SDCBP/MDA-9/Syntenin regulates protective autophagy in GSCs under anoikis conditions. This process is mediated through phosphorylation of the anti-apoptotic protein BCL2 accompanied with suppression of high levels of autophagic proteins (ATG5, LAMP1, LC3B) through EGFR signaling. SDCBP/MDA-9/Syntenin-mediated regulation of BCL2 and EGFR phosphorylation is achieved through PTK2/FAK and PRKC/PKC signaling. When SDCBP/MDA-9/Syntenin is absent, this protective mechanism is deregulated, leading to highly elevated and sustained levels of autophagy and consequently decreased cell survival. Our recent paper reveals a novel functional link between SDCBP/MDA-9/Syntenin expression and protective autophagy in GSCs. These new insights into SDCBP/MDA-9/Syntenin-mediated regulation and maintenance of GSCs present leads for developing innovative combinatorial cancer therapies.     

Gene expression changes in an animal melanoma model correlate with aggressiveness of human melanoma metastases

Metastasis is the deadliest phase of cancer progression. Experimental models using immunodeficient mice have been used to gain insights into the mechanisms of metastasis. We report here the identification of a "metastasis aggressiveness gene expression signature" derived using human melanoma cells selected based on their metastatic potentials in a xenotransplant metastasis model. Comparison with expression data from human melanoma patients shows that this metastasis gene signature correlates with the aggressiveness of melanoma metastases in human patients. Many genes encoding secreted and membrane proteins are included in the signature, suggesting the importance of tumor-microenvironment interactions during metastasis.     

Local and systemic inhibition of lung tumor growth after nanoparticle-mediated mda-7/IL-24 gene delivery

The human melanoma differentiation associated gene-7 (mda-7), also known as interleukin-24 (IL-24), is a novel gene with tumor suppressor, antiangiogenic, and cytokine properties. In vitro adenovirus-mediated gene transfer of the human mda-7/IL-24 gene (Ad-mda-7) results in ubiquitous growth suppression of human cancer cells with minimal toxicity to normal cells. Intratumoral administration of Ad-mda-7 to lung tumor xenografts results in growth suppression via induction of apoptosis and antiangiogenic mechanisms. Although these results are encouraging, one limitation of this approach is that its locoregional clinical application-systemic delivery of adenoviruses for treatment of disseminated cancer is not feasible at the present time. An alternative approach that is suitable for systemic application is non-viral gene delivery. We recently demonstrated that DOTAP:cholesterol (DOTAP:Chol) nanoparticles effectively deliver tumor suppressor genes to primary and disseminated lung tumors. In the present study, therefore, we evaluated nanoparticle-mediated delivery of the human mda-7/IL-24 gene to primary and disseminated lung tumors in vivo. We demonstrate that DOTAP:Chol efficiently delivers the mda-7/IL-24 gene to human lung tumor xenografts, resulting in suppression of tumor growth. Growth-inhibitory effects were observed in both primary (P=0.001) and metastatic lung tumors (P=0.02). Furthermore, tumor vascularization was reduced in mda-7/IL-24-treated tumors. Finally, growth was also inhibited in murine syngenic tumors treated with DOTAP:Chol-mda-7 nanoparticles (P=0.01). This is the first report demonstrating (1) systemic therapeutic effects of mda-7/IL-24 in lung cancer, and (2) antitumor effects of human mda-7 in syngeneic cancer models. Our findings are important for the development of mda-7/IL-24 treatments for primary and disseminated cancers.     

Human uveal melanoma cells produce macrophage migration-inhibitory factor to prevent lysis by NK cells

Human uveal melanoma arises in an immune privileged ocular environment in which both adaptive and innate immune effector mechanisms are suppressed. Uveal melanoma is the most common intraocular tumor in adults and is derived from tissues in the eye that produce macrophage migration-inhibitory factor (MIF), a cytokine that has recently been demonstrated to produce immediate inhibition of NK cell-mediated lytic activity. Although NK cell-mediated lysis of uveal melanomas is inhibited in the eye, melanoma cells that disseminate from the eye are at risk for surveillance by NK cells. Moreover, uveal melanoma cells demonstrate a propensity to metastasize to the liver, an organ with one of the highest levels of NK activity in the body. Therefore, we speculated that uveal melanomas produced MIF as a means of escaping NK cell-mediated lysis. Accordingly, seven primary uveal melanoma cell lines and two cell lines derived from uveal melanoma metastases were examined for their production of MIF. MIF was detected in melanoma culture supernatants by both ELISA and the classical bioassay of macrophage migration inhibition. Melanoma-derived MIF inhibited NK cell-mediated lysis of YAC-1 and uveal melanoma cells. Cell lines derived from uveal melanoma metastases produced approximately twice as much biologically active MIF as cultures from primary uveal melanomas. Inhibition of NK cell-mediated killing by uveal melanoma-derived MIF was specifically inhibited in a dose-dependent manner by anti-MIF Ab. The results suggest that human uveal melanoma cells maintain a microenvironment of immune privilege by secreting active MIF that protects against NK cell-mediated killing.     

Distinguishing Tumor and Stromal Sources of MicroRNAs Linked to Metastasis in Cutaneous Melanoma

MicroRNA (miRNA) dysregulation in cancer causes changes in gene expression programs regulating tumor progression and metastasis. Candidate metastasis suppressor miRNA are often identified by differential expression in primary tumors compared to metastases. Here, we performed comprehensive analysis of miRNA expression in The Cancer Genome Atlas (TCGA) skin cutaneous melanoma (SKCM) tumors (97 primary, 350 metastatic), and identified candidate metastasis-suppressor miRNAs. Differential expression analysis revealed miRNA significantly downregulated in metastatic tumors, including miR-205, miR-203, miR-200a-c, and miR-141. Furthermore, sequential feature selection and classification analysis identified miR-205 and miR-203 as the miRNA best able to discriminate between primary and metastatic tumors. However, cell-type enrichment analysis revealed that gene expression signatures for epithelial cells, including keratinocytes and sebocytes, were present in primary tumors and significantly correlated with expression of the candidate metastasis-suppressor miRNA. Examination of miRNA expression in cell lines revealed that candidate metastasis-suppressor miRNA identified in the SKCM tumors, were largely absent in melanoma cells or melanocytes, and highly restricted to keratinocytes and other epithelial cell types. Indeed, the differences in stromal cell composition between primary and metastatic tumor tissues is the main basis for identification of differential miRNA that were previously classified as metastasis-suppressor miRNAs. We conclude that future studies must consider tumor-intrinsic and stromal sources of miRNA in their workflow to identify bone fide metastasis-suppressor miRNA in cutaneous melanoma and other cancers.     

Analysis of monosomy‐3 in immunomagnetically isolated circulating melanoma cells in uveal melanoma patients

Monosomy‐3 in primary uveal melanoma (UM) is associated with a high risk of metastasis and mortality. Although circulating melanoma cells (CMC) can be found in most UM patients, only approximately 50% of the patients develop metastases. We utilized a novel immuno‐FISH assay to detect chromosome‐3 in intact CMC isolated by dual immunomagnetic enrichment. Circulating melanoma cells were detected in 91% of the patients (n = 44) with primary non‐metastatic UM, of which 58% were positive for monosomy‐3. The monosomy‐3 status of CMC corresponded to the monosomy‐3 status of the primary tumor in 10 of the 11 patients where this could be tested. Monosomy‐3 in the CMC was associated with an advanced tumor stage (P = 0.046) and was detected in all four patients who developed metastasis within the follow‐up period of 4 yr. This non‐invasive technique may enable the identification of UM patients at risk for metastasis particularly when a primary tumor specimen is unavailable.